A correlation has been made between the levels of one isoaccepting lysyl-tRNA, lys-tRNA4, and the ability of mammalian cells to undergo cell division. The inhibition of cell division leads to the build-up of different lys-tRNA species, which we have proposed to be undermodified intermediates in a lys-tRNA4 biosynthetic pathway. To confirm this pathway, we are sequencing the intermediates and developing in vitro assays for the various modification enzymes. Five different isoaccepting lys-tRNA species have been isolated and sequenced. Two new modified bases are present in lys-tRNA4 in the anticodon loop. One of these also is present in lys-tRNA2, a precursor to tRNA4lys, whereas the other is unique to tRNA4lys. Using extracts from rapidly dividing cells, we have been able to modify lys-tRNA2 and lys-tRNA5 to tRNA4lys in vitro. In one of these cases, we have been able to sequence the product of the reaction showing the addition of one of the tRNA4lys-specific modified bases.